The forces that drive clonal expansion of a precancerous cell, and the barriers that normally prevent it, are little understood. This Exploratory Grant project develops key resources for studying this problem. For skin cancer, the earliest known lesions are keratinocyte clones carrying p53 mutations. We find that clonal expansion of these mutant cells requires sustained UVB, but not additional mutations. Neighboring stem cell compartments normally act as a barrier to clonal expansion, but UVB allows p53-mutant keratinocytes to breach this barrier. By creating a knock-in mouse carrying a fluorescent P53-mRFP1 fusion protein, we will be able, for the first time, to dynamically observe the origin, expansion, and regression of p53-mutated clones in real time. A second approach, potentially applicable to human skin, visualizes clones by topically applying fluorescent peptides that tightly bind P53. These methods will be used in the second year to directly observe quantized jumping of mutant cells from one stem cell compartment to another, investigate the precursor-product relation of clones to precancers, and determine whether clonal expansion can be modified genetically using topical RNAi. In the future, we will ask: Does clonal expansion create new stem cells? Does a clone progress through actinic keratosis to carcinoma? Can UVA drive clonal expansion by reducing intercellular communication? Do clones regress by squaming? Do the regression-resistant clones arise from stem cells? Do rare mutant cells persist long after visible lesions have regressed? Can skin screening of RNAi libraries reveal genes affecting clone expansion? Thus, these technical advances should open up the field of clonal expansion.